plac8.2 Antibody, Biotin conjugated

What is PLAC8.2 and how does it differ from other PLAC8 family proteins?

PLAC8.2 is a member of the PLAC8 protein family found in zebrafish (Danio rerio), with the UniProt ID A2VD52. It represents one of the zebrafish orthologs of the placenta-specific gene 8 protein found in mammals. The PLAC8 family proteins are involved in various biological processes including organ development and tumorigenesis . Unlike mammalian PLAC8, which is approximately 12.5 kilodaltons in mass, the zebrafish PLAC8.2 (also known as Zgc:158845 protein) has distinct structural characteristics specific to teleost fish. PLAC8 proteins are expressed in various immune cells at different levels, with particularly high expression observed in Th1 CD4 T-cells compared to other T-cell subsets . The zebrafish ortholog maintains some conserved functions while exhibiting species-specific roles in development.

What are the key specifications of commercially available PLAC8.2 Antibody with Biotin conjugation?

The biotin-conjugated PLAC8.2 antibody is a polyclonal antibody raised in rabbits against a peptide sequence from zebrafish Zgc:158845 protein (amino acids 5-19) . The antibody has been purified using Protein G chromatography with a purity exceeding 95%. It is supplied in liquid form, containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4) . This antibody has been validated specifically for ELISA applications with zebrafish samples, though cross-reactivity with other species should be experimentally determined. The antibody's biotin conjugation enables high sensitivity detection schemes using streptavidin-based detection systems.

How does the structure and function of PLAC8.2 in zebrafish compare to human PLAC8?

While both human PLAC8 and zebrafish PLAC8.2 belong to the same gene family, they exhibit some structural and functional differences reflective of evolutionary divergence. Human PLAC8 (also known as placenta specific 8, C15, onzin, or PNAS-144) is a 12.5 kDa protein involved in immune cell function and cancer processes . Zebrafish PLAC8.2 shares conserved domains but has adapted functions relevant to teleost biology. The human PLAC8 is known to regulate inflammation by suppressing pro-inflammatory cytokines through enhancement of autophagy , while zebrafish PLAC8.2 functions are still being elucidated. In research applications, understanding these comparative aspects is crucial when using zebrafish as a model for human disease studies.

What are the optimal protocols for using PLAC8.2 Antibody, Biotin conjugated in ELISA assays?

For ELISA applications with PLAC8.2 antibody (biotin conjugated), researchers should follow this optimized protocol:

• Antigen Coating: Coat ELISA plates with recombinant PLAC8.2 protein or zebrafish tissue lysate at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block with 2-5% BSA in PBS for 1-2 hours at room temperature.

• Primary Antibody: Apply serially diluted test antibodies in blocking buffer for 1-2 hours at room temperature.

• Detection Antibody: Add the biotin-conjugated PLAC8.2 antibody at a dilution of 1:500 to 1:2000 in blocking buffer for 1 hour at room temperature .

• Streptavidin-HRP: Apply streptavidin-HRP conjugate (1:5000-1:10000) for 30-60 minutes.

• Visualization: Develop with TMB substrate and read absorbance at 450nm after stopping the reaction with 2N H₂SO₄.

When optimizing, perform a checkerboard titration to determine the ideal antibody concentration that provides the best signal-to-noise ratio. Include proper negative controls (non-specific rabbit IgG-biotin) and positive controls (known PLAC8-reactive samples).

How can PLAC8.2 Antibody be incorporated into proximity-dependent biotin labeling techniques?

Proximity-dependent biotin labeling techniques like BioID or APEX can be enhanced by incorporating PLAC8.2 antibody as follows:

• Modified BLITZ Approach: Adapt the Biotin Labelling In Tagged Zebrafish (BLITZ) methodology using PLAC8.2 antibody. This technique targets biotin ligase to GFP-labeled proteins of interest in transgenic zebrafish.

• Procedure Implementation:

  • Inject mRNA encoding GFP-tagged PLAC8.2 and BirA*-conjugated anti-GFP nanobody into zebrafish embryos

  • Administer biotin (50 μM) to living embryos at desired developmental stages

  • After fixation, detect biotinylated proteins using streptavidin-based visualization methods

  • For mass spectrometry analysis, extract and purify biotinylated proteins using streptavidin beads

• Comparative Analysis: Using pre-biotinylated PLAC8.2 antibody alongside the BLITZ method provides complementary data sets that can validate interaction partners of PLAC8.2 in vivo.

This technique is particularly valuable for identifying weak or transient PLAC8.2 protein interactions that traditional co-immunoprecipitation might miss, especially in specialized zebrafish tissues like immune cells or developing organs .

What troubleshooting strategies address common issues when using biotin-conjugated PLAC8.2 antibody?

When addressing persistent issues, consider validating the antibody reactivity using Western blot analysis of zebrafish tissue lysates to confirm specificity before proceeding with more complex applications.

How can PLAC8.2 antibody be utilized in comparative immunology studies across fish species?

Comparative immunology studies utilizing PLAC8.2 antibody can follow this methodological framework:

• Cross-reactivity Assessment:

  • Perform Western blot analyses against tissue lysates from multiple teleost species (e.g., medaka, stickleback, carp)

  • Create a phylogenetic cross-reactivity map showing antibody recognition patterns aligned with evolutionary distance

  • Validate epitope conservation through sequence alignment of the immunogen region (aa 5-19 of zebrafish PLAC8.2)

• Functional Conservation Analysis:

  • Employ immunohistochemistry to compare PLAC8.2 expression patterns in immune tissues across species

  • Correlate expression with immune challenge responses using standardized pathogen exposure protocols

  • Quantify relative expression levels using qPCR in parallel with immunological detection

• Evolutionary Significance Investigation:

  • Compare PLAC8.2 expression profiles between species with different immune system complexities

  • Assess correlation between PLAC8.2 expression/function and habitat-specific immune challenges

  • Determine if PLAC8.2 represents a conserved or divergent component of teleost immune function

This approach provides valuable insights into the evolution of immune-related proteins across fish species and helps establish zebrafish as a model for studying conserved immune mechanisms with potential relevance to human health .

What are the considerations for multiplex immunofluorescence involving biotin-conjugated PLAC8.2 antibody?

When designing multiplex immunofluorescence experiments with biotin-conjugated PLAC8.2 antibody, researchers should consider these critical factors:

• Signal Amplification Strategy:

  • Use streptavidin conjugated to spectrally distinct fluorophores (Alexa Fluor 488, 555, 647)

  • Consider tyramide signal amplification for low-abundance PLAC8.2 detection

  • Implement sequential detection if using multiple biotin-conjugated antibodies

• Panel Design Considerations:

  • Select companion antibodies raised in species other than rabbit to avoid cross-reactivity

  • Account for spectral overlap when choosing fluorophores for multi-color imaging

  • Include appropriate controls for autofluorescence (unstained tissue) and non-specific binding (isotype controls)

• Tissue-Specific Optimization:

  • For zebrafish tissues, optimize fixation protocols (4% PFA for 2-24h depending on tissue size)

  • Determine optimal antigen retrieval methods specific to zebrafish tissues

  • Adjust antibody concentrations based on PLAC8.2 expression levels in different tissues

• Data Acquisition and Analysis:

  • Use spectral unmixing algorithms for closely overlapping fluorophores

  • Implement colocalization analysis to quantify PLAC8.2 association with other proteins

  • Apply machine learning approaches for unbiased classification of cell types based on marker expression

Successful multiplex imaging allows simultaneous visualization of PLAC8.2 with other immune markers to characterize complex cellular interactions in zebrafish tissues that cannot be captured through single-marker studies.

How does PLAC8.2 expression correlate with immune cell activation states in zebrafish models?

Current research indicates that PLAC8.2 expression dynamics in zebrafish immune cells follow these patterns:

• Developmental Expression Profile:

  • PLAC8.2 expression emerges during hematopoietic specification in zebrafish embryos

  • Expression levels increase significantly during thymus development and T-cell maturation

  • Differential expression occurs between developing myeloid and lymphoid lineages

• Activation-Dependent Regulation:

  • Similar to mammalian PLAC8, zebrafish PLAC8.2 shows differential expression across T-cell subsets

  • Expression appears to increase following immune stimulation with PAMPs (pathogen-associated molecular patterns)

  • Preliminary data suggests potential roles in the resolution phase of inflammation rather than initiation

• Cell Type-Specific Expression Patterns:

  • Predominant expression in zebrafish myeloid lineage cells

  • Variable expression in T-cell populations depending on activation status

  • Lower expression in B-cells compared to other leukocyte populations

This expression pattern analysis, enabled by the biotin-conjugated PLAC8.2 antibody, provides insights into potential functional parallels between zebrafish PLAC8.2 and human PLAC8, particularly in contexts like inflammation regulation and immune cell differentiation pathways .

How can PLAC8.2 antibody be integrated into single-cell analysis workflows?

Integration of biotin-conjugated PLAC8.2 antibody into single-cell analysis requires specialized protocols:

• Single-Cell Antibody-Based Cytometry:

  • Dissociate zebrafish tissues using optimized protocols (0.25% trypsin-EDTA, gentleMACS)

  • Stain single-cell suspensions with biotin-PLAC8.2 antibody (1:100-1:500 dilution)

  • Detect with streptavidin-fluorophore conjugates compatible with cytometry panels

  • Include viability dyes and suitable gating strategies for accurate cell type identification

• CITE-seq and Related Technologies:

  • Conjugate oligonucleotide barcodes to PLAC8.2 antibody for cellular indexing of transcriptomes

  • Use the antibody at 0.5-2 μg per million cells in the CITE-seq antibody cocktail

  • Process samples following established CITE-seq protocols with appropriate sequencing depth

  • Analyze data for correlation between PLAC8.2 protein levels and transcriptional profiles

• Imaging Mass Cytometry Applications:

  • Label PLAC8.2 antibody with rare earth metals through biotin-streptavidin bridging

  • Apply to tissue sections following IMC protocols with optimized antibody concentration

  • Include panel design considerations to avoid signal spillover between channels

  • Implement neighborhood analysis to identify spatial relationships between PLAC8.2+ cells

These approaches enable unprecedented resolution in understanding PLAC8.2 distribution across zebrafish immune cell populations and reveal heterogeneity not apparent in bulk analyses.

What considerations are important when using PLAC8.2 antibody in proteomics workflows?

For proteomics applications involving PLAC8.2 antibody, researchers should consider these methodological aspects:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Utilize the biotin-conjugated antibody with streptavidin beads for efficient capture

  • Implement stringent washing (high salt, detergents) to reduce non-specific binding

  • Consider crosslinking strategies to capture transient interactions

  • Include appropriate controls (IgG-biotin, competing peptide) to filter out false positives

• Proximity-Dependent Biotin Labeling:

  • Adapt the BLITZ methodology for zebrafish models as described in recent literature

  • Engineer fusion constructs combining PLAC8.2 with promiscuous biotin ligases (BioID2, TurboID)

  • Optimize biotin supplementation (50 μM) and labeling time for zebrafish systems

  • Apply appropriate extraction conditions to retain membrane-associated interactions

• Quantitative Interaction Proteomics:

  • Implement SILAC or TMT labeling for quantitative comparison across conditions

  • Validate key interactions through reciprocal pulldowns and orthogonal methods

  • Apply bioinformatic filtering using CRAPome databases to exclude common contaminants

  • Construct interaction networks specific to zebrafish PLAC8.2 with functional annotation

This proteomics approach has proven valuable for defining protein interaction networks in vivo for related proteins in zebrafish muscle tissue , suggesting similar success could be achieved for PLAC8.2 in immune contexts.

How does post-translational modification of PLAC8.2 affect antibody recognition and protein function?

Research on PLAC8.2 post-translational modifications (PTMs) has revealed important considerations for antibody-based studies:

• Known and Predicted PTMs:

  • Phosphorylation at conserved serine/threonine residues affects protein localization

  • Potential ubiquitination sites regulate protein turnover and stability

  • Predicted N-myristoylation may influence membrane association properties

• Impact on Antibody Recognition:

  • The immunogen region (aa 5-19) may contain modification sites that affect epitope accessibility

  • Phosphorylation-dependent epitope masking can occur under certain cellular activation states

  • Denaturation during sample preparation may expose epitopes normally obstructed by PTMs

• Functional Consequences:

  • Phosphorylation appears to regulate PLAC8.2 translocation between cytoplasmic and nuclear compartments

  • PTM patterns change during immune cell activation, suggesting regulatory importance

  • Interspecies conservation of modification sites indicates functional significance

• Methodological Approaches:

  • Phosphatase treatment of samples prior to antibody application can assess modification dependence

  • Combined use of modification-specific and pan-PLAC8.2 antibodies provides functional insights

  • Mass spectrometry analysis of immunoprecipitated PLAC8.2 can map the PTM landscape

Understanding these PTM dynamics is essential for accurate interpretation of PLAC8.2 antibody data in functional studies, particularly when examining immune cell activation states.

What controls are essential when validating PLAC8.2 antibody specificity in zebrafish models?

A comprehensive validation strategy for PLAC8.2 antibody should include these essential controls:

• Genetic Controls:

  • CRISPR/Cas9-generated PLAC8.2 knockout zebrafish as negative controls

  • PLAC8.2 overexpression systems as positive controls

  • Morpholino knockdown with titrated doses for partial depletion controls

• Biochemical Validation:

  • Pre-absorption with immunizing peptide (aa 5-19) to confirm epitope specificity

  • Western blot analysis showing a single band at the expected molecular weight

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Cross-Reactivity Assessment:

  • Testing against related zebrafish proteins (other PLAC8 family members)

  • Evaluation in tissues known to be negative for PLAC8.2 expression

  • Species cross-reactivity testing if using in comparative studies

• Application-Specific Controls:

  • For immunohistochemistry: Isotype controls at matching concentrations

  • For flow cytometry: Fluorescence-minus-one (FMO) controls

  • For proximity labeling: BirA* expression without fusion partner

This validation framework ensures that experimental observations can be confidently attributed to specific detection of PLAC8.2 rather than technical artifacts or cross-reactivity.

How do different fixation and permeabilization protocols affect PLAC8.2 antibody performance?

The choice of fixation and permeabilization methods significantly impacts PLAC8.2 antibody performance:

Permeabilization Considerations:

• For whole zebrafish embryos/larvae: 0.5-1% Triton X-100 in PBS (2-4 hours)

• For tissue sections: 0.1-0.3% Triton X-100 or 0.05% Tween-20 (10-30 minutes)

• For cultured cells: 0.1% Triton X-100 or 0.1% Saponin (5-15 minutes)

When optimizing protocols, progressive testing of fixation duration and permeabilization strength is recommended, as PLAC8.2 epitope accessibility may vary depending on developmental stage and tissue context.

What are the considerations for quantitative analysis of PLAC8.2 expression across different zebrafish tissues?

Quantitative analysis of PLAC8.2 expression requires standardized approaches:

• Sample Standardization:

  • Age-matched specimens (specific hours post-fertilization or adult age)

  • Consistent husbandry conditions to minimize environmental variables

  • Standardized tissue dissection or cell isolation protocols

• Quantitative Methods Comparison:

  • Western blot densitometry: Suitable for bulk tissue analysis with 15-20% precision

  • qPCR: Measures transcript levels but may not correlate with protein expression

  • Flow cytometry: Provides single-cell resolution with 5-10% coefficient of variation

  • Imaging analysis: Offers spatial information but requires careful normalization

• Reference Standards:

  • Internal loading controls (β-actin, GAPDH) for Western blot normalization

  • Spike-in controls of known concentration for absolute quantification

  • Multi-tissue calibration curves for inter-tissue comparisons

• Technical Considerations:

  • Signal linearity determination through titration experiments

  • Consistent imaging parameters for fluorescence quantification

  • Background subtraction methodology standardization

  • Statistical power analysis to determine appropriate sample sizes

This quantitative framework enables reliable comparison of PLAC8.2 expression across different experimental conditions, developmental stages, and tissue types, providing a foundation for functional studies.